Project Summary Gliomas are the most frequent form of primary brain tumors. Survival rates are low, and the often dismal outcome highlights our poor understanding of the defining features of glioma cells and their resistance to radiation or chemotherapy. Glioma contains a small population of glioma stem cells (GSCs) capable of asymmetric self-renewal and multi-lineage differentiation. The clinically relevant features of GSCs include high tumorigenic potency and therapeutic resistance. Previous studies failed to identify definitive cell surface markers of tumor-propagating GCSs, and no markers are known for therapeutic resistance. We propose to test the hypothesis that quiescence confers to a subgroup of GSCs high tumorigenic potency and therapy- resistance. To test this hypothesis, we designed an innovative kinetic analysis that enables in vivo tracking of the proliferative history of glioma cells and that allows sorting glioma cells into slow- and fast-dividing subgroups. This is achieved by genetic engineering of human glioma-derived GSCs (hGSCs) with a doxycycline-inducible Histone2B-GFP label. A pulse-and-chase study will identify fast-dividing tumor cells as GFP- due to dilution, whereas quiescent cells will remain GFP+. Subsequent stem cell cultures will then select tumor stem cells from these two groups for further studies. Such a kinetic analysis is advantageous in capturing the dynamic in vivo behaviors of glioma cells. In Aim 1, we will track three hGSC lines for their in vivo proliferative behaviors in xenotransplants at different time points. After tumor growth, GFPhigh and GFPlow subgroups will be analyzed for their proportion, aggregation patterns, dissemination distance, expression of neural stem cell or differentiation markers, and spatial relationship to known neural stem cell niches. In Aim 2, we will sort the GFPhigh and GFPlow subgroups and subject them to neural stem cell culture conditions to isolate gliomaspheres. The GSCfast and GSCslow will be compared for their self-renewal capacity and differentiation potential, as well as their migratory and tumor-forming capabilities. Gene expression profiling studies will identify unique molecular features. GSCs will also undergo two more passages as gliomaspheres to assess whether prior in vivo proliferation history leaves a proliferation memory that impacts future cellular behaviors. In Aim 3, we will apply radiation therapy (XRT) to test the hypothesis that quiescence confers GSCs with radiation-resistance. XRT-resistant glioma cells will be examined for their GFP labeling, and then be isolated for molecular characterization. This paradigm also offers an opportunity to study in vivo proliferative behaviors of XRT-resistant glioma cells in the post-XRT microenvironment. In summary, we combine innovative genetic engineering, human brain tumors, stem cell biology, and molecular studies to understand the impact of in vivo proliferative history on subsequent cellular behaviors of glioma stem cells as well as its link to tumorigenic potency and therapeutic resistance. Our kinetic studies based on a dynamic parameter of in vivo cell division will address the intratumoral functional heterogeneity of high-grade glioma and the underlying causes.